Preliminary Development Of Nucleic Lateral Flow Immunoassay And Dual Real-time PCR Assay Of Brucella

Brucellosis is an ancient zoonotic disease caused by the species of brucella,leads to severe clinical symptoms in multiple systems and poses a serious threat to health in both human and livestock and relative international trade as well.Current diagnose standard of brucellosis in China marked traditional immunological tests such as SAT and RBPT as prevailing methods to assist clinical diagnosis,which are easy and fast to perform but subjective when read the result,and also inaccurate because of its cross-reaction with some other bacterium.Thus its essential to improve the veracity,sensibility and speed of the methods.This study primarily established nucleic-acid lateral flow immunochromatographic method and dual fluorescence quantitative PCR aimed brucella virulence factors that can be handy to applied in hospitals and fields to assist screening and diagnose of brucellosis.In this study,the target fragments such as OMP10,OMP19,OMP25,OMP31,BCSP31 of brucella common virulence factors were designed and screened at the first place,and selected a pair of primer of the BCSP3 1 protein as the target.Marked both 5’ends of both forward and reverse primers with biotin and digoxin.Perform regular PCR as the upstream reaches of the lateral flow test strip.The optimum concentration of primers（10nM）and annealing temperature（55℃）were adjusted,and the reaction system were decided:premix PCR buffer 25μl,FR primer each 2.5μl,DNA template 5μl,DEPC water fill to 50μl.Colloidal gold was prepared by trisodium citrate reduction method.Selected the material of sample pad and binding pad.After optimizing the procedure to label antibodies with colloidal gold and the compound of sample buffer,the preprocessing method of the pads,then assembled the test strips.Dilute the template DNA into a series gradient and perform regular PCR to reveal the sensitivity of the experiments.Specificity tests were carried out by amplifying negative reference bacteria DNA as templates.Multiple sets of repeated tests were performed in several different batches to verify repeatability.The detection limit of the system is 10-2ng/μl,and shows no cross-reaction with salmonella,E.coli and proteus etc.Accelerated experiments at 37℃ by 24h and 72h showed satisfying stability and repeatability of operation between batches.In the second part of the study,based on the screened primers in the former part,4 pairs of primers（IS711,16SrRNA,OMP25,and BCSP31）were selected by assessing Ct value,amplification and dissolution curves.Recombinant plasmids were constructed for these 4 pairs of primers respectively.Dilute the plasmids into series gradient as template standards to prepare calibration curves.By comparing the slope with the theoretical value and the amplification efficiency,two pairs of primers（IS711,OMP25）were selected as the primer combination for the dual rt-PCR.The concentrations and ratios of primers,the amounts and ratios of template DNA,and the qPCR setup were adjusted and optimized to obtain the optimal system for the dual reaction.Dual calibration curve was prepared by combining two template standards at the same dilution.The slope was very close to the theoretical value,and the amplification efficiency was satisfying.Dilute the recombinant plasmid to a certain low gradient to perform the sensitivity test of the dual system.Apply the negative reference bacteria DNA as templates for specificity.The detection limit of this system is 10 copies and shows no cross-reaction with salmonella,E.coli and proteus etc.Operation repeatability between batches is preferable and system is stable.In this study,two methods for brucellosis detection were primarily developed.Nucleic acid lateral flow test is easy and convenient to operate,fast and clear to read,which indicates its value to be applied into grass-roots detection and speed up the diagnosis.Calibration curve for dual-fluorescence quantitative PCR method was constructed that offers a relative quantification of nucleic acid loading in the clinical sample.The combination of 2 primers also lifted check-out rate that can also help clinical diagnosis.